Background

Lignin’s valorization plays a critical role in refining the bioresource. Considering that the β-aryl ether linkage (β-O-4 bond) accounts for 50–70% of lignin chemical linkage between aromatic rings, the hydrolase of lignin β-aryl ether linkage, especially the β-etherase, provided a promising way for the lignin depolymerization and valorization. As a result, it is essential to develop the effective high-throughput methods for screening the mutant library of β-etherase from directed evolution.

Results

Based on the enzymatic mechanism of β-O-4 bond’s cleavage by β-etherase, the LigF was selected as the model to study high-throughput method by GSH assay for screening the mutant library of β-etherase from directed evolution. After the primary study with purified LigF and cell lysate, the GSH assay was used to screen mutant library of β-etherase. The study on screening the mutant library with about 600 colonies indicated that the selected transformants all have one or two mutated sites in the gene sequence of LigF, and the activities from GSH assay of most selected transformants were the same as their activities from HPLC assay.

Conclusions

The results from the high-throughput screening of mutant library demonstrated that GSH assay could be applied to screen β-etherase mutant from directed evolution.

中文翻译：

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谷胱甘肽（GSH）分析从定向进化中高通量筛选木质素β-芳基醚键水解酶的方法

背景

木质素的增值作用在提炼生物资源中起着至关重要的作用。考虑到β-芳基醚键（β-O-4键）占芳香环之间木质素化学键的50-70％，木质素β-芳基醚键的水解酶，特别是β-醚酶，提供了一种有前途的方法用于木质素的解聚和增值。结果，必须开发有效的高通量方法，以从定向进化中筛选β-醚酶的突变文库。

结果

基于β-醚酶裂解β-O-4键的酶促机理，选择LigF作为模型，通过GSH法研究高通量方法，从定向进化中筛选β-醚酶突变体文库。在使用纯化的LigF和细胞裂解液进行初步研究后，使用GSH分析法筛选β-醚酶的突变文库。筛选具有约600个菌落的突变体文库的研究表明，所选择的转化体在LigF的基因序列中均具有一个或两个突变位点，并且大多数所选择的转化体的GSH测定的活性与HPLC测定的活性相同。

结论

突变体文库的高通量筛选结果表明，GSH法可用于从定向进化中筛选β-醚酶突变体。